Dosage and administration for preventing cardiotoxicity in treatment with ERBB2-targeted immunoliposomes comprising anthracycline chemotherapeutic agents

ABSTRACT

Methods for determining dosage of HER2-targeted anthracycline-containing immunoliposomes are disclosed, as are methods of treating cancer patients with HER2-positive tumors using dosages so determined. Upon administration, the dosages share the low cardiotoxicity profile of standard dosages of non-immunoliposomal (untargeted), anthracycline-containing liposomes.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of PCT International PatentApplication No. PCT/US2011/063623, filed Dec. 6, 2011, which claims thebenefit of and priority to U.S. Provisional Patent Application Nos.61/420,225, filed Dec. 6, 2010; 61/420,688, filed Dec. 7, 2010; and61/449,602, filed Mar. 4, 2011. The contents of each of the foregoingapplications are incorporated herein by reference in their entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

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REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

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BACKGROUND OF THE INVENTION

Anthracyclines have been an effective backbone of cancer therapies fordecades. Despite consistent clinical benefit observed withanthracycline-based regimens in breast cancer, significant toxicitiessuch as acute and/or chronic cardiac dysfunction associated with suchtreatment have limited more expansive therapeutic use. While liposomaldoxorubicin formulations have succeeded in reducing cardiotoxicity tosome extent, they have failed to demonstrate clear-cut efficacyadvantages and can involve other toxicities such as palmar-plantarerythrodysesthesia (hand foot syndrome). In an effort to improve uponefficacy of currently available anthracyclines, a new immunoliposomalformulation, MM-302, has been prepared that targets doxorubicin to HER2(ErbB2)-overexpressing tumor cells. Antibody fragments that bind to HER2without blocking HER2-mediated signaling are coupled to the outersurface of pegylated liposomal doxorubicin.

Doxorubicin (dox) is an anthracyline chemotherapeutic agent used totreat a variety of cancers. The use of doxorubicin is dose-limited bythe cardiotoxicity of the drug. In order to address this problem,doxorubicin has been formulated as a pegylated liposomal preparation.Liposomal encapsulation of drugs enables delivery of potent cytotoxicdrugs with an improved therapeutic index or therapeutic window.doxorubicin HCl liposome injection (DOXIL®) is a pegylatedliposome-encapsulated (liposomal) form of doxorubicin. DOXIL is acommercial form of pegylated liposomal doxorubicin (PLD). DOXIL altersthe tissue distribution and pharmacokinetic profile of doxorubicin. Useof DOXIL results in a significantly lower rate of left ventricularcardiac dysfunction and symptomatic congestive heart failure as comparedto therapy with free doxorubicin, both alone and in combination withtrastuzumab in anthracycline-naïve and previously treated patients.DOXIL® is approved for use to treat Kaposi's sarcoma, ovarian cancer,and multiple myeloma. Doxorubicin HCl liposome injection is also sold asCAELYX®.

Immunoliposomes are antibody (typically antibody fragment) targetedliposomes that provide advantages over non-immunoliposomal preparationsbecause they are selectively internalized by cells bearing cell surfaceantigens targeted by the antibody. Such antibodies and immunoliposomesare described, for example, in the following U.S. patents and patentapplications: US 2010-0068255, U.S. Pat. Nos. 6,214,388, 7,135,177, and7,507,407 (“Immunoliposomes that optimize internalization into targetcells”); U.S. Pat. No. 6,210,707 (“Methods of forming protein-linkedlipidic microparticles and compositions thereof”); U.S. Pat. No.7,022,336 (“Methods for attaching protein to lipidic microparticles withhigh efficiency”) and US 2008-0108135 and U.S. Pat. No. 7,244,826(“Internalizing ErbB2 antibodies.”). The following US and internationalpatents and patent applications describe assays, cell lines, and relatedtechnologies that are relevant to this disclosure: U.S. Pat. No.7,846,440 (“Antibodies against ErbB3 and uses thereof”) and U.S. Ser.No. 12/757,801, PCT/US2009/040259, and PCT/US2009/60721 (“Human SerumAlbumin Linkers and Conjugates Thereof”).

Immunoliposomes targeting ErbB2 (HER2) can be prepared in accordancewith the foregoing patent disclosures. Such HER2 targetedimmunoliposomes include MM-302, which comprises the F5 anti-HER2antibody fragment and contains doxorubicin. MM-302 contains 45 copies ofmammalian-derived F5-scFv (anti-HER2) per liposome. The F5-scFv wasselected for its ability to internalize while not affecting HER2signaling. Characterization of the F5-scFv indicates that it does notcross react with mouse, rat or rabbit HER2 and does not interfere withHER2 signaling in the free scFv form. Because cardiomyocytes are knownto express HER2, concerns have been expressed regarding the potentialcardiotoxicity of MM-302 and related HER2-targeted immunoliposomes.

Dosage and Administration of Commercially Available LiposomalDoxorubicin:

DOXIL® (doxorubicin HCl liposome injection) is an exemplary liposomalanthracycline chemotherapeutic drug. DOXIL is typically administeredintravenously at a dose indicated in mg/m² and characterized asdoxorubicin HCl equivalent (dox equiv., meaning the total mass ofdoxorubicin in each dose). Each dose is typically administered at aninterval measured in weeks, to yield a dosage of x mg/m² (dox equiv.)every y weeks. The first liposomal doxorubicin dose is typicallyadministered at an initial rate of 1 mg/min to minimize the risk ofinfusion-related reactions. If no infusion-related adverse reactions areobserved, the infusion rate is typically increased to complete theadministration of the drug over one hour.

Patients with Ovarian Cancer:

DOXIL is typically administered to ovarian cancer patients intravenouslyat a dose of 50 mg/m² dox equiv. The patient is typically dosed onceevery 4 weeks, for as long as the patient does not progress, shows noevidence of cardiotoxicity and continues to tolerate treatment. Aminimum of 4 courses is recommended because median time to response inclinical trials was 4 months. To manage adverse reactions such ashand-foot syndrome (HFS), stomatitis, or hematologic toxicity the dosesmay be delayed or reduced. Pretreatment with or concomitant use ofantiemetics should be considered.

Patients with AIDS-Related Kaposi's Sarcoma (KS):

DOXIL is typically administered to KS patients intravenously at a doseof 20 mg/m² (dox equiv.). In KS patients the dose is typically repeatedonce every three weeks, for as long as patients respond satisfactorilyand tolerate treatment.

Patients with Multiple Myeloma:

To treat patients with multiple myeloma, DOXIL is administered withVELCADE® (bortezomib). Bortezomib is administered at a dose of 1.3 mg/m²as intravenous bolus on days 1, 4, 8 and 11, every three weeks. DOXIL istypically administered to these patients at a dose of 30 mg/m² as a 1-hrintravenous infusion following each day 4 bortezomib administration.Patients are typically treated for up to 8 cycles until diseaseprogression or the occurrence of unacceptable toxicity.

HERCEPTIN® (trastuzumab) is a therapeutic anti-HER2 antibody that isvery widely used to treat HER2 overexpressing tumors. A keydosage-limiting effect of trastuzumab is cardiotoxicity. Cardiomyocytesare known to express HER2, and trastuzumab-mediated cardiotoxicity isgenerally accepted as being caused by damage to HER2-expressingcardiomyocytes resulting from trastuzumab binding to thecardiomyocyte-expressed HER2—see, e.g., Hysing J and Wist E,“Cardiotoxic Effects of Trastuzumab,”. Tidsskr Nor Laegeforen, 2011 Nov.15; 131(22):2239-2241. Anthracycline drugs such as doxorubicin are knownto exert dose-limiting cardiotoxic effects, which are considered a majorlimitation in their use—see, e.g., Sawyer et al., “Mechanisms ofAnthracycline Cardiac Injury: Can we identify strategies forcardio-protection?” Prog Cardiovasc Dis., 2010 September-October;53(2):105-13.

Doxorubicin-induced cardiac damage is irreversible, resulting in acuteinjury and also damage that can manifest itself years after treatment.Exposure to cumulative concentrations of doxorubicin above 550 mg/m²increases the potential for cardiomyopathy and heart failure. Thedevelopment of HER2-directed therapy for the treatment of HER2-positivebreast cancer has led to the investigation of the clinical benefit ofthe combination of doxorubicin and trastuzumab. The clinical efficacy ofdoxorubicin plus trastuzumab was superior to that of paclitaxel plustrastuzumab; however, there was an increased incidence of cardiactoxicity observed on the doxorubicin plus trastuzumab arm of the study,and the combination was not approved for marketing. The clinical benefitof anthracycline-based therapy, specifically in HER2-positive breastcancer, remains controversial.

Liposomal encapsulation of drugs has enabled delivery of potentcytotoxic drugs with an improved therapeutic index. Pegylated liposomaldoxorubicin (PLD) alters the tissue distribution and pharmacokineticprofile of doxorubicin. PLD has demonstrated a significantly lower rateof left ventricular cardiac dysfunction and symptomatic congestive heartfailure as compared to therapy with conventional doxorubicin, alone andin combination with trastuzumab in anthracycline-naïve and previouslytreated patients. A proposed mechanism for the reduced cardiotoxicity ofPLD is that its greater size relative to conventional doxorubicinprevents it from crossing the endothelial barrier in the heart, therebyminimizing doxorubicin exposure to heart tissue.

MM-302 is a HER2-targeted, pegylated liposome designed to deliverdoxorubicin directly to HER2-overexpressing cancers. HER2-targeted PLDdeposits in tumors through the enhanced permeability and retentioneffect similar to PLD. In the tumor microenvironment, targetingHER2-overexpressing cells with HER2-targeted PLD results in superiorefficacy relative to PLD in preclinical models. During the developmentof MM-302, concern was expressed by regulatory authorities that due toits HER2-targeting, MM-302 would deliver cardiotoxic doxorubicindirectly to cardiomyocytes, resulting in increased cardiotoxicitycompared to doxorubicin HCl liposome injection, and reduced dosages ofMM-302 were suggested to avoid such life-threatening toxicities.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods to determine safe doses and tosafely use anti-HER2 immunoliposomal anthracyclins to treatHER2-expressing cancers, e.g., without increased risk of cardiotoxicityas compared to doxorubicin HCl liposome injection (DOXIL), and providesother advantages.

It has now been discovered that anti-ErbB2 targeted,anthracycline-containing immunoliposomes, e.g., MM-302, are not any morecardiotoxic than doxorubicin HCl liposome injection (DOXIL®), and can bedosed using exactly the same dosages, (i.e., dose and administrationamounts and schedules) as used for doxorubicin HCl liposome injectionwithout any increase in cardiotoxicity risk or decrease in efficacy.Furthermore, it has now been demonstrated that MM-302 can be effectivelytargeted to cells expressing 200,000 or more ErbB2 (HER2) receptors percell in vitro and in vivo, indicating that it can be used to treatpatients with HER2-overexpressing tumors that are either HER2 “3+”(e.g., by HERCEPTEST®), HER2 FISH+ (fluorescence in situ hybridizationfor HER2 gene amplification) or HER2 “2+” (e.g., by HERCEPTEST).

Therefore, disclosed herein are methods for determining a safe andeffective dosage for use in treating a human cancer patient byadministration of anthracycline-comprising anti-HER2 immunoliposomes,the patient being diagnosed with a cancer characterized by expression ofHER2 receptor, the methods comprising determining a first dosage, such adosage indicating a dose magnitude and frequency of dosing, for apatient diagnosed with a cancer characterized by expression of HER2receptor, the first dosage being for a liposomal anthracyclinechemotherapeutic agent that does not comprise an immunoliposome, whichdosage is determined to provide to the patient a safe and effectiveamount of the liposomal anthracycline chemotherapeutic agent; anddetermining a dosage for the administration of theanthracycline-comprising anti-HER2 immunoliposomes, a plurality of whichimmunoliposomes is each bearing a plurality of anti-HER2 antibodymolecules on its surface and each containing the anthracyclinechemotherapeutic agent, where the safe and effective dosage for theadministration of the anthracycline-comprising anti-HER2 immunoliposomesis the first dosage.

Also disclosed are methods of treating a human cancer patient byadministration of anthracycline-comprising anti-HER2 immunoliposomes,the methods comprising determining a first dosage, such a dosageindicating a dose magnitude and frequency of dosing, for a patientdiagnosed with a cancer characterized by expression of HER2 receptor,the first dosage being for a liposomal anthracycline chemotherapeuticagent that does not comprise an immunoliposome, which dosage isdetermined to provide to the patient a safe and effective amount of theliposomal formulation, and administering anthracycline-comprisinganti-HER2 immunoliposomes, a plurality of which immunoliposomes is eachbearing a plurality of anti-HER2 antibody molecules on its surface andeach containing the anthracycline chemotherapeutic agent, where theanthracycline-comprising anti-HER2 immunoliposomes are administered tothe patient at the first dosage.

In certain aspects the anthracycline is doxorubicin. In other aspectsthe liposomal anthracycline chemotherapeutic agent that does notcomprise an immunoliposome is doxorubicin HCl liposome injection and theHER2-targeted immunoliposomes are MM-302. In others, the cancer isbreast cancer, Kaposi's sarcoma, ovarian cancer, or multiple myeloma. Inyet other aspects, the first dosage is 50 mg/m², 40 mg/m², 30 mg/m², 20mg/m², or 10 mg/m² every two weeks or every three weeks or every fourweeks. In other aspects the cancer characterized by expression of HER2receptor is further characterized as being HER2²⁺, HER2³⁺, or HER2 FISHpositive. In others the cancer characterized by expression of ErbB2receptor is further characterized as expressing an average of at least200,000 cell surface ErbB2 receptors per cell. In yet others, theadministration of the immunoliposomes at the first dosage is effectiveto treat the cancer and in others the administration of theimmunoliposomes at the first dosage does not result in increasedcardiotoxicity as compared to administration at the first dosage of theliposomal anthracycline chemotherapeutic agent that does not comprise animmunoliposome. In other aspects, the administration of theimmunoliposomes to the patient at the first dosage results in a peakconcentration of the immunoliposomes in the patient's bloodstream, andtreating human cardiomyocytes in vitro by culturing in medium comprisingthe immunoliposomes at about the peak concentration does not reduce, orreduces by no more than 5%, heregulin-stimulated increase of pERK orpAKT in the cultured cardiomyocytes as compared to in control humancardiomyocytes cultured in medium free of the immunoliposomes. In otheraspects the immunoliposome concentration in the patient's bloodstream ismeasured as a serum immunoliposome concentration. In yet other aspectseach of the HER2 immunoliposomes bears on its surface, on average, 45anti-HER2 antibody molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Effect of Her2 Levels on Uptake: Multiple cell lines expressingvarious levels of HER2 were treated with 15 μg/ml of MM-302 (A) anduntargeted pegylated liposomal dox (UT-PLD) (B) for 2 h and totalcellular doxorubicin was quantified by HPLC. The y-axes representfemtograms dox per cell (left) and liposomes per cell (right) and thex-axes represent the number of HER2 receptors per cell (log scale).Mouse tumor 4T1 cells (C) and endogenously low HER2 expressing HeLacells (D) were transfected with human HER2 to generate stable cloneswith varying levels of expression. Individual clones (represented bytriangles, circles, or squares) were treated with F5-targeted liposomescontaining a fluorescent marker (DiI5-F5-PL), and total binding/uptakewas determined by FACS.

FIG. 2. (A) HER2-overexpressing BT474-M3 cells were treated with 15μg/ml of MM-302 (circle), PLD (square), and free doxorubicin (triangle)for the indicated times (x-axis, in min.). Total cellular doxorubicinwas quantified by HPLC (y-axis, femtograms/cell). (B) Nucleardoxorubicin delivery was quantified by high content microscopy (y-axis,signal per cell above background) 24 h following the indicatedincubation times (x-axis, in min) (C) The anti-tumor activity of MM-302and PLD were compared in a BT474-M3 orthotopic breast cancer model. BothMM-302 (square) and PLD (triangle) significantly inhibited tumor growthcompared to control (circle) (t-test at day 55; p<0.0001). MM-302resulted in a stronger inhibition of tumor growth relative to PLD(t-test at day 55; p=0.0310). The y-axis indicates tumor volume in mm³and the x-axis indicates days after inoculation. FIG. 2D showspharmacokinetics of MM-302 and UT-PLD in BT474-M3 xenograftsadministered with 3 mg/kg or 6 mg/kg (dox equiv) at q7d. The y-axis isμg/ml dox in plasma and the x-axis is time in hours.

FIG. 3. Role of HER2 Levels in vivo: Mice bearing BT474-M3 xenografttumors in the mammary fat pad were injected with Dil5-labeled MM-302(Dil5-F5-PLD) or UT-PLD (Dil5-UT-PL). A tumor single cell suspension isprepared and stained with FITC-HER2 antibodies.Dil5-positive-HER2-positive cells were determined by FACS. (A) A graphshowing HER2 level (receptors per cell, x-axis) as a function ofliposome binding to tumor cells (percent cells positive, y-axis). (B) Agraph demonstrating the heterogeneity of HER2 expression on a singlecell basis as measured in tumor tissue sections.

FIG. 4. Uptake of MM-302 (circle), PLD (square) and doxorubicin(triangle) was measured in human cardiomyocytes. Embryonic stemcell-derived (ESCd) (A) and induced pluripotent stem cell-derived (iPSd)(B) cardiomyocytes were treated with 15 μg/ml of MM-302, PLD and freedoxorubicin for the indicated times. Total cellular doxorubicin wasquantified by HPLC. The y-axes represents uptake in femtograms/cell andthe x-axes represent incubation time in min. (C) Cell viability: ESCdcardiomyocytes were treated for 3 h with drug at the indicatedconcentrations and incubated for an additional 24 h with fresh media andcell viability was assessed. The y-axis represents cell viability as %compared to control and the x-axis represents concentration in μg/ml.(D) Cell viability: iPSd cardiomyocytes were treated with the indicatedconcentration of free doxorubicin (circle), PLD (square) or MM-302(triangle) for 24 hours. The supernatant was collected and a PrestoBlue®cell viability assay was performed on the remaining cells. All valueswere normalized to the untreated population. (E) A human Troponin IELISA was performed on the supernatant collected in (D). The untreatedline (dotted line) represents the value of soluble Troponin I detectablein untreated wells. All values are normalized against dilutions of asupplied standard.

FIG. 5. ESCd cardiomyocytes were treated for 3 h with MM-302 (circle),PLD (square), and free doxorubicin (triangle) at the indicatedconcentrations and then incubated for an additional 24 h with freshmedia. Cells were stained and imaged using high-content microscopy.Single cell intensity for each stain was quantified and represented asthe mean relative intensity of individual cells. Cells were stained forthe DNA damage marker gamma-H2AX (A). In addition, cells were stainedfor the cell stress proteins phospho-p53 (B) and phospho-HSP27 (C), andthe cleaved form of the apoptosis protein PARP (cPARP) (D). The y-axesrepresent relative intensity and the x-axes represent concentration inμg/ml.

FIG. 6. (A) F5 scFv alone, or F5 scFv bound to empty (i.e. withoutencapsulated dox) liposomes (“F5 lipo”—liposomes equivalent to MM-302but not containing doxorubicin) minimally decrease basal pERK levels anddo not decrease heregulin stimulated pERK levels in iPS-derived humancardiomyocytes, while Herceptin® (trastuzumab) decreases basal levels toa greater extent and both trastuzumab and lapatinib decrease heregulinstimulated levels to a greater extent than do F5 scFv or F5 lipo. (B)None of the tested agents changed basal pAKT levels in these cells andthat trastuzumab and lapatinib both decrease heregulin stimulated levelsto a significantly greater extent than do F5 scFv or F5 lipo.

FIG. 7. (A) The biodistribution of MM-302 (square), PLD (triangle) andfree doxorubicin (circle) was studied. NCI-N87 tumor bearing mice(n=4/time point/group) were given a single dose (3 mg/kg) of eitherdoxorubicin, MM-302 or PLD. Mice were sacrificed at 0.5, 4 and 24 h postinjection and the doxorubicin accumulation in heart tissue (A), tumortissue (B) and paw (C) was quantified by HPLC. (D) MM-302 induces lowernuclear doxorubicin accumulation in heart tissue compared to freedoxorubicin, and comparable to PLD. Nu/nu mice were injectedintravenously with MM-302-DiI5, PLD-DiI5, and free doxorubicin at 3mg/kg (dox equiv.). At the designated time points, hearts were collectedfor the preparation of cryosections to analyze the microdistribution ofliposomes and doxorubicin. FITC-lectin was injected to visualizefunctional/perfused blood vessels. Heart sections were counterstainedwith Hoechst and imaged by confocal fluorescence microscopy (40×magnification). Doxorubicin positive nuclei are shown. Doxorubicinpositive nuclei are visible in the free doxorubicin treated samples at0.5 h and 4 h and not in MM-302 treated samples (see “merge panels”).(E) A higher magnification (2×) of the overlay of the nuclei anddoxorubicin signal of the above fields for the 0.5 h time points fordoxorubicin and MM-302 is shown. (F) The percent of doxorubicin positivenuclei was quantified using Definiens® Developer XD™ (F).

FIG. 8: Modeling: A computational model was developed and calibrated onliterature and in-house data for free and liposomal doxorubicin. It isapplied to understand the competing kinetic processes that determinedrug concentration and exposure for liposomal versus free doxorubicin invarious tissues. (A) PK and Biodistribution: In contrast with freedoxorubicin, liposomal delivery results in a much longer circulationtime. (B) Tissue Deposition: The model is able to capture typicalliposome deposition data in mouse. (C) Microdistribution: Kineticmodeling is able to provide a framework for understanding the role ofHER2 expression in MM-302 uptake and doxorubicin cellular trafficking.

DETAILED DESCRIPTION OF THE INVENTION

An MM-302 liposome is a unilamellar lipid bilayer vesicle ofapproximately 75-110 nm in diameter that encapsulates an aqueous spacewhich contains doxorubicin in a gelated or precipitated state. The lipidmembrane is composed of phosphatidylcholine, cholesterol, and apolyethyleneglycol-derivatized phosphatidylethanolamine in the amount ofapproximately one PEG molecule for 200 phospholipid molecules, of whichapproximately one PEG chain for each 1780 phospholipid molecules bearsat its end an F5 single-chain Fv antibody fragment that binds to HER2.MM-302 liposomes are prepared from HSPC (Hydrogenated soyphosphatidylcholine): Cholesterol (plant-derived): PEG-DSPE(polyethylene glycol-disteroylphosphoethanolamine) at a molar ratio of3:2:0.3. The total HSPC lipid concentration of MM-302 is about 40mmol/L. MM-302 contains about 10 mmol/L of lipid, and about 2 mg/mL ofdoxorubicin. MM-302 comprises 1.8-2.2 mg/mL of doxorubicin in liposomesthat contain 0.16-0.30 mg/mL DSPE-PEG-F5 (prepared as described in U.S.Pat. No. 6,210,707). F5 is an anti-ErbB2 (HER2) scFv antibody fragment(encoded by ATCC plasmid deposit designation PTA-7843). MM-302 liposomescomprise 130-170 g doxorubicin/mol phospholipid and 12-22 gF5-PEG-DSPE/mol phospholipid. MM-302 is formulated in sterile 10 mM/Lhistidine-HCl as a buffer (pH 6.5), and 10% sucrose to maintainisotonicity. MM-302 liposomes are loaded using pre-loaded ammoniumsulfate

MM-302 Dosing

Dose 1 Dose 2 Dose 3 Dose 4 Dose 5 Every 10 mg/m² 20 mg/m² 30 mg/m² 40mg/m² 50 mg/m² week Every 10 mg/m² 20 mg/m² 30 mg/m² 40 mg/m² 50 mg/m²two weeks Every 10 mg/m² 20 mg/m² 30 mg/m² 40 mg/m² 50 mg/m² three weeksEvery 10 mg/m² 20 mg/m² 30 mg/m² 40 mg/m² 50 mg/m² four weeks Every 10mg/m² 20 mg/m² 30 mg/m² 40 mg/m² 50 mg/m² five weeks“mg/m²” indicates mg of doxorubicin (formulated as MM-302) per squaremeter of body surface area of the patient. For breast cancer, dose 3, 4,or 5 is preferred. For Kaposi's sarcoma dose 1, 2, or 3 is preferred,for ovarian cancer, dose 3, 4, or 5 is preferred and for multiplemyeloma dose 2, 3, 4, or 5 is preferred. Dosing regimens may vary inpatients with solid tumors that are “early” (pre-metastatic, e.g.,adjuvant breast cancer) as compared to “advanced” (metastatic tumors).Preferred tumors are those in which the tumor cells overexpress HER2. Atumor that overexpresses HER2 is one that is identified as being HER2“3+” or HER2 “2+” by HercepTest™, or HER2 FISH+ by fluorescence in situhybridization. Alternatively, a preferred tumor that overexpresses HER2is one that expresses an average of 200,000 or more receptors per cell,as quantified by the methods described in the Examples.MM-302 Therapy of Advanced Breast Cancer

MM-302 is administered once every 4 weeks by intravenous (IV) infusionover 60 minutes at 8, 16, 30, 40, or 50 mg/m² to patients with locallyadvanced/unresectable or metastatic advanced breast cancer thatoverexpresses HER2 as determined by FISH or by IHC or by determinationof the average number of HER2 receptors per cell. Patients should haveadequate bone marrow reserves as evidenced by: 1) absolute neutrophilcount (ANC)≧1,500/μL; 2) platelet count≧100,000/μL and 3) hemoglobin≧9g/dL (Transfusions allowed). Patients should have adequate hepaticfunction as evidenced by: 1) serum total bilirubin≦1.5×ULN and 2)Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) andAlkaline Phosphatase (ALP) normal or up to 2.5×upper limit of normal(ULN; 5×ULN is acceptable for ALP if liver metastases and/or bonemetastases are present). Patients should have adequate renal function asevidenced by a serum creatinine≦1.5×ULN. Patients should be recoveredfrom any clinically relevant toxic effects of any prior surgery,radiotherapy or other therapy intended for the treatment of breastcancer. Women of childbearing potential as well as fertile men and theirpartners must be warned to abstain from sexual intercourse or to use aneffective form of contraception during treatment and for 90 daysfollowing the last dose of MM-302. Patients should have adequate cardiacfunction as evidenced by a measured left ventricular ejection fractionof ≧50% by ECHO or MUGA within approximately 30 days of treatment.Patients who are pregnant or lactating and those with NYHA Class III orIV congestive heart failure or left ventricular ejection fraction(LVEF)<50%, or a prolonged QTc interval (≧460 ms), are preferably not betreated with MM-302.

The following Examples are merely illustrative and should not beconstrued as limiting the scope of this disclosure in any way as manyvariations and equivalents will become apparent to those skilled in theart upon reading the present disclosure.

EXAMPLES Materials and Methods Used in these Examples

-   Materials: Doxorubicin is from SIGMA-ALDRICH, Inc. (St. Louis, Mo.).    FITC-conjugated lectin (lycopersicon esculentum (tomato) lectin, Cat    #FL-1171) is purchased from Vector Laboratories, Inc. (Burlingame,    Calif.). Acetic acid, Methanol, and Acetonitrile are from EMD    Chemicals Inc. (Gibbstown, N.J.). Water and Trifluoroacetic Acid    (TFA) are from J. T. Baker (Phillipsburg, N.J.). HOECHST 33342    trihydrochloride trihydrate, ProLong Gold®, and DiIC18(5)-DS (DiI5)    are from Invitrogen (Carlsbad, Calif.). Cholesterol and    1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene    glycol)-2000] (ammonium salt) (PEG-DSPE) are from Avanti Polar    Lipids Inc. Hydrogenated soy phosphatidylcholine (HSPC) is from    Lipoid (Newark, N.J.). RPMI is from Lonza (Walkersville, Md.), Fetal    Bovine Serum (FBS) is from Tissue Culture Biologicals and penicillin    G/streptomycin sulphate mixture is from GIBCO (Invitrogen).-   Preparation of immunoliposomes: Liposomes are prepared and loaded    with doxorubicin using an ammonium sulfate gradient as previously    described (Kirpotin et. al., Cancer Res. 2006; 66:6732-40; Park et    al., Clin Cancer Res. 2002; 8:1172-81). The lipid components are    HSPC, cholesterol, and PEG-DSPE (3:2:0.3, mol:mol:mol). The    anti-ErbB2 (F5)-PEG-DSPE conjugate is prepared and inserted into the    liposome to form immunoliposomes as reported by Nellis et al.,    (Biotechnol Prog. 2005; 21:205-20; Biotechnol Prog. 2005;    21:221-32). The DiI-5-labelled liposomes, MM-302-DiI5 and PLD-DiI5,    are prepared as above with the difference that the DiIC18(5)-DS    (DiI5) dye is solubilized with the lipid components at a    concentration of 0.3 mol % of total phospholipid. In all cases    unloaded free doxorubicin is removed using a Sephadex® G-75 size    exclusion column eluted with Hepes buffered saline (pH 6.5).    F5-lipo-DiI5 is prepared in a similar fashion as above but without    doxorubicin, and incorporating an aqueous solution of HEPES buffered    saline (pH 6.5).-   Tumor cell culture: BT474-M3 cells (see Noble, Cancer Chemother.    Pharmacol. 2009 64:741-51), are HER2-overexpressing human breast    cancer cells. BT474-M3 cells are grown in RPMI medium containing 10%    FBS and 1% penicillin G/streptomycin sulphate. Embryonic stem    cell-derived (ESCd) cardiomyocytes are obtained from P. W. Zandstra,    Institute of Biomaterials and Biomedical Engineering, University of    Toronto, Toronto, Ontario, Canada; (Bauwens et al., Tissue Eng    Part A. 2011 Apr. 25, PMID 21417693). These cells have been shown to    express appropriate cellular markers of cardiomyocytes such as LIM    domain homeobox gene Isl-1, Troponin T, and Myosin Light Chain 2c.    The percentage of Troponin T positive cells is determined following    differentiation. Batches containing less than 70% positive for    Troponin T are discarded. Induced pluripotent stem cell-derived    (iPSd) cells are obtained from Cell Dynamics International and are    handled per the manufacturer's protocol.-   Xenograft Studies: 5-7-week-old female nude mice are purchased from    Charles River Laboratories or Taconic Farms, Inc (NCr nude mice).    Unless otherwise indicated, mice are inoculated with BT474-M3 breast    cancer cells or NCI-N87 gastric cancer cells (NCI-DTP, 10×10⁶ cells    in 100 μl RPMI) into the right dorsal flank of the mice    (subcutaneous injection, s.c.). When the tumors reach an average    volume of ˜200 mm³, studies are performed as described below.-   Testing of tumor HER2 levels: Homozygous NCr nude mice are    inoculated with 15×10⁶ BT474-M3 cells in the mammary fat pad 2nd    from the top right hand side. BT474-M3 tumors are injected with a    single dose of 4 mg/ml fluorescently-labeled (with DiI5 as described    below) HER2-targeted or untargeted liposomes without doxorubicin.    Twenty-four hours later, the tumors are excised and dissociated by    mechanical and enzymatic means. After surface staining with    anti-HER2 cells are analyzed by flow cytometry on a FACSCalibur™    instrument (BD Biosciences). The flow cytometry dataset is analyzed    for the relationship between HER2 surface expression levels and    uptake of liposomes above a given threshold by plotting the overall    percentage of cells with elevated liposome content in narrowly gated    cell subsets defined by increasing HER2 signals.-   Single cell distribution of cell surface HER2: BT474-M3 tumor    xenograft tissue is stained with an anti-HER2 antibody and    counterstained with DAPI. The slide is imaged with an Aperio®    Scanscope® at 20× magnification and the image is analyzed. The    intensity of the HER2 membrane staining is quantified on a    single-cell basis as the (mean of the inner border of the HER2    layer)+(mean of the outer border of the HER2 layer).-   Efficacy study: Mice are randomized into three treatment groups    (n=7/group) based on an average tumor volume from mice that receive    PBS (control), MM-302 or PLD, dosed at 3 mg/kg (q7d, n=3 total    doses). Tumors are measured twice/week with a caliper. Tumor volumes    are calculated using the formula: width²×length×0.52. Mice are    weighed twice/week to monitor weight loss.-   Uptake of liposomes in HER2-expressing cell lines: Multiple cell    lines expressing various levels of HER2 are treated with 15 μg/ml of    MM-302 or PLD for 2 h and total cellular doxorubicin is quantified    by HPLC. Murine 4T1 breast cancer cells and human HeLa cervical    cancer cells are obtained from the ATCC and propagated according to    ATCC recommendations. Cells are characterized for human HER2    expression by flow cytometry using a commercial anti-HER2 antibody    (BD Biosciences #340552). This antibody does not cross-react with    murine HER2 but detects human HER2. A neomycin-selectable expression    vector encoding human HER2 is obtained from GeneCopeia (Z2866). 4T1    and HeLa cells are transfected with this construct using the    non-lipid polymer transfection reagent MegaTran® 1.0 (Origene)    according to the manufacturer's instructions. Transfected cells are    selected with 400-500 μg/ml Geneticin/Neomycin (Invitrogen).    Surviving cells are allowed to expand under reduced    Geneticin/Neomycin concentrations (100 μg/ml) and are sorted on a BD    Biosciences FACSAria™ instrument to obtain enriched cell populations    with human HER2 expression exceeding those observed in parental HeLa    cells. The sort-enriched cells are then sub-cloned by limited    dilutions, and colonies are ranked by HER2 surface levels to obtain    representative populations of 4T1 and HeLa cells that express    different ranges of HER2. Fluorescent intensity of HER2 surface    staining measured by flow cytometry is compared to staining with the    same antibody bound to Quantum™ Simply Cellular® anti-mouse IgG    microspheres (Bangs Laboratories #815) according to the    manufacturer's instructions to calculate the number of HER2 surface    receptors of the cells.-   Uptake of liposomes in cardiomyocytes: iPSd cardiomyocytes are    plated per the manufacturer's instructions (Cell Dynamics    International Cat #CMC-100-110-001) in a 24-well tissue culture    plate at 250,000 cells/well. Two days later, the 0.5 ml of media in    the wells is removed and replaced with 0.5 ml of 15.0 μg/ml (dox    equiv.) of MM-302, PLD or free doxorubicin. The plates are swirled    in a “figure 8 fashion” 20 times to maximize exposure of the cells    to the liposomes. Cells are incubated with MM-302, PLD or free    doxorubicin for the indicated time period after which the media are    removed and the cells are washed once with 0.5 ml of PBS. The PBS is    removed and 0.5 ml of 0.05% trypsin is added to each well. The cells    are monitored, and once detachment begins, 0.5 ml of medium    containing FBS is added to each well to inactivate the trypsin. The    cells are collected and placed in microcentrifuge tubes. The cells    are spun at 1,500 rpm for 5 minutes at 4° C. The cell pellet is    resuspended by vortexing and pipetting in 0.5 ml of 1.0% acetic acid    in methanol and placed at −80° C. for 1 hour to extract the    doxorubicin. The microcentrifuge tube containing the resuspended    cell pellet is spun at 10,000 rpm for 10 minutes in the cold room,    and 450 μl of the supernatant is transferred to a HPLC tube. Samples    are run on the HPLC machine and concentration per sample is    determined by relating values to a doxorubicin standard curve.-   Biodistribution of liposomes: Mice are randomized into 4 groups that    received a single intravenous (i.v.) dose of either PBS,    doxorubicin, MM-302-DiI5 or PLD-DiI5 (all at 3 mg/kg), respectively.    Mice (n=4/time point/group) are sacrificed at 0.5, 4, 24 and 96 h    (doxorubicin) or 168 h (MM-302-DiI5 and PLD-DiI5) after the single    dose. Five minutes before sacrificing, mice are injected i.v. with    100 μl of FITC-lectin, to label the vasculature.-   HPLC quantification of doxorubicin: Heart tissues are weighed and    disaggregated with 1 mL H₂O using a TissueLyser™ (Qiagen) for 3 min.    100 μl of the homogenate is then transferred into a new tube and 900    μl of 1% acetic acid/methanol is added. For the cultured cells,    cells are treated with drug, as described above, trypsinized and    lysed using 1.0% acetic acid in methanol. Lysates are vortexed for    10 sec. and placed at −80° C. for 1 h. Samples are spun at room    temperature (RT) for 10 min at 10,000 RPM. Supernatants and    doxorubicin standards are analyzed by HPLC (Dionex) using a C18    reverse phase column (Synergi™ POLAR-RP® 80 Å, 250×4.60 mm 4 μm    column). Doxorubicin is eluted running a gradient from 30%    acetonitrile; 70% 0.1% trifluoroacetic acid (TFA)/H₂O to 55%    acetonitrile; 45% 0.1% TFA/H20 during a 7 min span at a flow rate of    1.0 ml/min. The doxorubicin peak is detected at 6.5 min using an    in-line fluorescence detector excited at 485 nm, and emitting at 590    nm. The extraction efficiency of doxorubicin from the heart tissue    was 83% as determined by a control heart spiked with a known amount    of doxorubicin.-   Confocal microscopy and image analysis of heart snap-frozen    sections: 10 μm-thick heart sections are air-dried for 30 min at RT,    counterstained with Hoechst® 33342 diluted 1:10,000 in mounting    media (ProLong® Gold, Invitrogen) and mounted. Slides are imaged on    a LSM 510 Zeiss® confocal microscope equipped with Enterprise (351,    364 nm), Argon (458, 477, 488, 514 nm), HeNe1 (543 nm) and HeNe2    (594 nm) lasers with a Plan-Neofluar® 40×/1.3 oil DIC objective.    Image analysis and quantification of nuclear doxorubicin is carried    out using Definiens® Developer XD™ (Definiens, Parsippany, N.J.).    Nuclei are segmented in the Hoechst channel. Doxorubicin positive    nuclei are segmented in the doxorubicin channel. The percentage of    doxorubicin positive nuclei is quantified as a ratio of the number    of objects in the doxorubicin channel divided by the total nuclei    objects in the Hoechst channel.-   Receptor quantification: Stem cell-derived cardiomyocytes are    trypsinized, washed, and HER2 levels are determined as described    above under “Uptake of liposomes in HER2-expressing cell lines.”-   Viability: ESCd cardiomyocytes are treated for 3 h at the indicated    concentrations of MM-302, PLD and doxorubicin. Cells are washed    twice with PBS, fresh medium is added and the cells are incubated    for an additional 24 h. Cell viability is assessed using    CellTiter-Glo® from Promega (Madison, Wis.) and the percent of    viable cells is determined relative to the untreated population.-   Troponin I ELISA: 15,000 iPSd (iCELL®) cardiomyocytes (Cellular    Dynamics International, Madison Wis.) cells are plated per the    manufacturer's protocol. Cells are treated for 24 h with the    indicated concentrations of free doxorubicin, PLD or MM-302. The    supernatant is collected and analyzed using a human Troponin I ELISA    (Catalog #: GWB-83A61F, Genway Biotech, San Diego Calif.) per the    manufacturer's protocol. A PrestoBlue® Cell Viability Assay (Catalog    #: A-13261, Invitrogen, Grand Island, N.Y.) is performed on the    remaining cells in 100 μl per the manufacturer's protocol.-   High-Content Analysis: Cardiomyocytes are treated as described    above. Cells are fixed using 3.7% formaldehyde, washed twice with    PBS containing 0.1% Tween-20 (PBS-T), and permeabilized with    methanol. Cells are blocked using a 1:1 mixture of LI-COR® Odyssey®    Blocking Buffer (Lincoln, Nebr.) and PBS-T for 1 h at room    temperature (RT). Cells are stained with a 1:400 dilution of the    indicated primary antibody from Cell Signaling Technology (Beverly,    Mass.) and incubated shaking at 4° C. overnight. Cells are washed    and incubated with a 1:2,000 dilution of the fluorescently labeled    secondary antibody for 1 h at RT. Cells are stained with a 1:10,000    dilution of Hoechst 33342 and 1:1,000 dilution of Whole Cell Stain    from Pierce Protein Research Products (Rockford, Ill.) for 30 min at    RT to allow visualization of DNA and the whole cell, respectively.    Plates are scanned using the Applied Precision Instruments    ArrayWorx® High Content Scanner (Issaquah, Wash.) with a 10×    objective for Hoechst 33342/Whole Cell Stain (460 nm), doxorubicin    (595 nm), and APC/DiI5 (657 nm). Images are analyzed using the    software ImageRail (as described in Millard et al., Nat Methods.    2011; 8:487-92). An intensity threshold is established for nuclear    and whole cell signals. This threshold is then applied to all images    and used to segment individual cells. Data is presented as the mean    pixel intensity for all cells in a given well for the indicated    channel.-   Signaling in cardiomyocytes: iPS-derived cardiomyocytes (iCell™    iPS-derived human cardiomyocytes—Cellular Dynamics International    (CDI), Madison, Wis.—CDI # CMC-100-010-001, Lot 1258680) are    cultured using iCell™ Plating Medium (CDI #CMM-100-110-005, Lot    1013740 and iCell™ Maintenance Medium (CDI #CMM-100-120-005,    Lot 1000305) and are exposed to components of MM-302. Levels of    phospho-AKT (pAKT) and phospho-ERK (pERK) in the cardiomyocytes are    then measured using immunostaining and high content microscopy.    Cells are pretreated for 24 hours with either trastuzumab,    lapatinib, or the MM-302 antibody (F5-scFv) and an MM-302 molecule    (liposome) not containing doxorubicin (F5-lipo) at an equivalent    concentration to 5.0 ug/ml of MM-302. Cells are stained and imaged    using high content microscopy as described above for (A) pAKT    and (B) pERK following a 10 minute stimulation with 10 nM and 5 nM    of heregulin, respectively. The following primary antibodies are    used at a 1:400 dilution in blocking buffer: pERK antibody—Cell    Signaling Technology (CST—Danvers Mass.)—Catalog 9106L    (Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10) Mouse mAb    #9106); pAKT antibody—CST—Catalog 4060L (Phospho-Akt (Ser473) (D9E)    XP™ Rabbit mAb #4060). Secondary antibodies are Anti-mouse IgG    (H+L), F(ab′)2 Fragment (Alexa Fluor® 647 Conjugate)—CST—Catalog    4410; Anti-rabbit IgG (H+L), F(ab′)2 Fragment (Alexa Fluor® 647    Conjugate)—CST—Catalog 4414. Whole cell stain and DNA stain    (Hoechst 33342) are used as described above. Images are analyzed and    individual cells are segmented on the basis of Hoechst 33342 nuclear    staining. Single cell signal intensity for each stain is quantified    and represented as the mean relative intensity of individual cells.

The results in the following Examples were obtained using the abovemethods or minor variations thereof. Cellular uptake studies in tumorcell lines expressing various levels of HER2 demonstrate that MM-302delivers significantly higher doxorubicin levels to HER2 over-expressingtumor cells compared to PLD as well as similar or higher levels thanhighly permeable free doxorubicin. However, in human cardiomyocytes,while free doxorubicin was again taken up at high levels, doxorubicinuptake was dramatically lower with both MM-302 and PLD. Pharmacokineticstudies in mice demonstrate that MM-302 has a similar half-life,clearance, and organ distribution compared to PLD. InHER2-overexpressing BT474 breast and NCI-N87 gastric tumor xenografts,MM-302 exhibits superior anti-tumor activity to both free anthracyclinesand PLD. Tumor microdistribution studies further suggest thatdifferences in the localization of doxorubicin in the tumor may beresponsible for the enhanced activity of MM-302 compared to freedoxorubicin and PLD.

Example 1 Correlation Between HER2 Expression and MM-302 Uptake In Vitro

The level of cell surface HER2 expression on multiple cell lines wasdetermined as described above. These same cell lines were then treatedwith 15 μg/ml of MM-302 (FIG. 1A, Table 1) or PLD (FIG. 1B, Table 2) for180 minutes, after which the cells were collected and the amount of cellassociated doxorubicin was quantified by HPLC. By plotting the number ofHER2 receptors per cell for each cell line vs. the quantity ofdoxorubicin present per cell in that same cell line following treatment,a relationship between increasing HER2 levels and increasing doxorubicinbecomes evident. Through this representation, there appears to be aninflection point at approximately 200,000 HER2 receptors per cell wherecells expressing greater than this number appear to have consistentlyhigher levels of cell associated doxorubicin. Taken together, theseresults support the specificity of MM-302, with high uptake by cellsexpressing levels of HER2 above the inflection point (such as HER2overexpressing cancers) and no-to-minimal uptake in cells expressinglevels of HER2 below the inflection point (such as cells in normaltissues, e.g., cardiomyocytes).

TABLE 1 HER2 levels vs MM-302 uptake FIG. HER2 fg 1A Cell line Source(#/cell) dox/cell A 4T1-clone- ATCC # CRL-2539 ™ 650,000 1,758.63 12W7 BADRr ATCC # HTB-22 ™ (MCF7 derivative) 40,792 26.47 C AdRr-Her2 ATCC #HTB-22 ™, stably transfected with HER2 722,000 519.04 D BT474-M3 Noble,Cancer Chemother. Pharmacol. 2009 64: 741-51 1,706,601 1,123.51 E Calu-3ATCC # HTB-55 ™ 1,196,976 84.06 F HCC1954 ATCC # CRL-2338 ™ 700,0001,174.60 G HeLa ATCC # CCL-2 ™ 123,713 31.61 H IGROV1 NCI 60-cell panelfrom NCI-DTP, DCTD TUMOR 158,418 51.54 REPOSITORY, Operated by CharlesRiver Laboratories, Inc. (NCI-DTP) I JIMT-1 DSMZ # ACC-589 850,000126.85 J MCF7 ATCC # HTB-22 74,745 17.52 K MCF7-c18 Gift from Dr.Christopher Bentz, Director, Cancer 1,031,247 531.31 and DevelopmentalTherapeutics Program, Buck Institute for Age Research, UCSF L MDA-MB-ATCC # HBT-27 ™ 371,731 112.79 361 M MDA-MB- ATCC # HBT-131 ™ 393,441185.51 453 N MKN-7 Health Science Research Resource Bank of the1,217,989 181.73 Japanese Health Sciences Foundation #JCRB1025 O NCI-N87ATCC # CRL-5822 ™ 1,233,479 366.10 P OVCAR8 NCI 60-cell panel fromNCI-DTP 53,272 99.79 Q OVCAR8- NCI 60-cell panel from NCI-DTP, stablytransfected 673,300 711.87 Her2 with HER2 R SkBr3 ATCC # HBT-30 ™1,315,512 228.68 S SKOV3 ATCC # HTB-77 ™ 1,377,661 600.81 T ZR75-1 ATCC# CRL-1500 ™ 199,132 30.32 (Results for MKN-45 cells were belowdetection level)

TABLE 2 HER2 levels vs PLD uptake FIG. HER2 fg 1B Cell Line Source(#/cell) dox/cell A 4T1-clone- ATCC # CRL-2539 ™ 650,000 13.54 12W7 BADRr ADR-RES (NCI-DTP) 40,792 23.24 D BT474-M3 Noble, Cancer Chemother.Pharmacol. 2009 64: 741-51 1,706,601 106.60 E Calu-3 ATCC # HTB-55 ™1,196,976 41.64 G HeLa ATCC # CCL-2 ™ 123,713 291.11 H IGROV1 NCI60-cell panel from NCI-DTP 158,418 34.94 I JIMT-1 DSMZ # ACC-589 850,000155.46 J MCF7 ATCC # HTB-22 74,745 13.57 K MCF7-c18 Gift from Dr.Christopher Bentz, Director, Cancer and 1,031,247 115.88 DevelopmentalTherapeutics Program, Buck Institute for Age Research, UCSF M MDA-MB-ATCC # HBT-131 ™ 393,441 59.97 453 N MKN-7 Health Science ResearchResource Bank of the Japanese Health 1,217,989 48.24 Sciences Foundation#JCRB1025 O NCI-N87 ATCC # CRL-5822 ™ 1,233,479 47.44 P OVCAR8 NCI60-cell panel from NCI-DTP 53,272 90.53 Q OVCAR8- NCI 60-cell panel fromNCI-DTP, stably transfected with HER2 673,300 37.00 Her2 S SKOV3 ATCC #HTB-77 ™ 1,377,661 43.83 T ZR75-1 ATCC # CRL-1500 ™ 199,132 26.10 UMKN-45 DSMZ # ACC-409 152,197 57.02 (Results for AdRr-Her2, HCC1954, andMDA-MB-361 cells were below detection level)

In order to quantify uptake into the different cell populations, MM-302was prepared to contain a red-fluorescent carbocyanine tracerDiIC18(5)-DS (Invitrogen D12730—abbreviated DiI5). DiI5 is a lipophilicfluorescent dye that intercalates into the lipid bilayer of the liposomeduring the extrusion process. 4T1-Her2 cell populations expressingdifferent ranges of human HER2 were incubated with 10 μg/mlfluorescently labeled MM-302 for 3 hrs, washed and incubated for anadditional 21 hrs. Cells were harvested, stained for cell surface humanHER2 and analyzed for both HER2 levels and liposome binding via flowcytometry. While the 4T1 cell line expresses murine HER2, MM-302 doesnot bind to the murine receptor. The figure shows that uptake of theseliposomes into 4T1 cells was strongly dependent on human HER2 expression(FIG. 1C) Similar results were obtained for populations of the HeLa celllines expressing different ranges of HER2 (FIG. 1D). These resultsfurther demonstrate that MM-302 is highly effective in binding cellswith high HER2 levels but has little or no binding to cells withrelatively lower HER2 protein expression.

Example 2 MM-302 is Effectively Internalized into HER2-OverexpressingTumor Cells and Significantly Inhibits Tumor Growth in a Xenograft Model

To determine levels of binding and internalization of MM-302 into HER2over-expressing tumor cells, BT474-M3 cells (1.7×10⁶ HER2/cell) wereincubated with MM-302, PLD or free doxorubicin at 15 μg/ml for up to 3 h(FIG. 2A). MM-302 was efficiently taken into tumor cells, as evidencedby total cell binding (FIG. 2A) and nuclear doxorubicin accumulation(FIG. 2B). By contrast, the untargeted analogue, PLD, did not show anyappreciable accumulation demonstrating the requirement of targeting toeffectively deliver liposomal doxorubicin in vitro. As a control, freedoxorubicin was shown to freely enter cells and accumulate in thenucleus. Results showed effective binding and internalization of MM-302(but not PLD) into HER2-overexpressing tumor cells.

The anti-tumor activity of MM-302 was evaluated in a breast cancerxenograft model. Mice were inoculated with BT474-M3 cells and when thetumor volumes reached an average of 250 mm³, treatment with PBS(control), MM-302 or PLD (both at 6 mg/kg dox equiv.) was started (q7d,n=3 doses). Both MM-302 and PLD significantly inhibited tumor growthrelative to control (t-test at day 55; p<0.0001). MM-302 resulted in astronger inhibition of tumor growth relative to PLD (t-test at day 55;p=0.0310) (FIG. 2C). At study termination, 3 complete regressions wereobserved with MM-302 and only 1 with PLD. MM-302 and PLD had similarpharmacokinetic profiles (FIG. 2D) indicating that the improved efficacywas as a result of HER2-targeting, rather than prolonged exposure.

Example 3 Impact of HER2 Levels on MM-302 Uptake In Vivo

Experiments were conducted to demonstrate HER2-mediated uptake of MM-302into target tumor cells from xenograft models compared with untargetedliposomal doxorubicin. Mice bearing BT474-M3 xenograft tumors in themammary fat pad were injected with Dil5-labeled MM-302 (Dil5-F5-PLD) orUT-PLD (Dil5-UT-PL). A tumor single cell suspension was prepared andstained with FITC-HER2 antibodies. Dil5-positive-HER2-positive cellswere determined by FACS. A distinct population of cells with elevateddoxorubicin levels was identified, indicating that liposomes had notjust been deposited in the tumor interstitial space, but had been takenup into the cells themselves (FIG. 3A). This was particularly evident incell samples derived from tumors treated with HER2-targeted liposomes.The percentage of cells with elevated liposome content began to rise incell subsets expressing on average 100,000 and 200,000 HER2 receptorsper cell. Untargeted liposomes did not show any preferential uptake intoHER2 positive cells. These results demonstrate that MM-302 uptake intumor cells in vivo is HER2-dependent and further support a level of atleast 100,000-200,000 HER2 receptors per cell necessary to allowsignificant binding and internalization of MM-302.

The distribution of HER2 membrane intensity was determined on asingle-cell basis in full tissue sections and is shown in FIG. 3B,representing the variability of expression in the tissue.

Example 4

Human Cardiomyocytes do not Express Sufficient HER2 Levels to ActivelyTake Up MM-302:

Human cardiomyocytes have been reported to express low levels of HER2,and therefore were thought to have potential for MM-302 uptake. ESCd andiPSd human cardiomyocytes were obtained to study the effect of MM-302 onhuman cardiac cells in vitro. HER2 receptor levels on cardiomyocyteswere determined by qFACS to be approximately 70,000 and 200,000receptors per cell in human ESCd and iPSd cardiomyocytes, respectively.These results are consistent with the reported low HER2 expression inhuman cardiac tissue (Fuchs et al., Breast Cancer Res Treat. 2003;82:23-8).

HER2 expression levels on normal and diseased human heart tissue weremeasured via quantitative immunohistochemistry. A human heart TissueMicroarray (TMA) was stained for HER2 and DAPI and the (Mean HER2intensity)/core was quantified with Definiens® software. A cell pelletarray with cell lines at different HER2 expression levels was stained asabove and the (Mean HER2 intensity)/core was quantified and plottedagainst the correspondent LOG (HER2 receptor #) to generate a standard.Based on the generated standard, the average HER2 receptor #/core forthe different human heart TMA cores was interpolated (Table 3).

TABLE 3 Interpolated HER2 Receptor Number ID Pathology Diagnosis HER2 #1 Chronic rheumatic valvular disease with calcification 40,000 2-pt1Chronic rheumatic valvular disease 38,000 2-pt2 Chronic rheumaticvalvular disease 38,000 2-pt3 Chronic rheumatic valvular disease 47,0002-pt4 Chronic rheumatic valvular disease 47,000 2-pt5 Chronic rheumaticvalvular disease 39,000 2-pt6 Chronic rheumatic valvular disease 38,0002-pt7 Chronic rheumatic valvular disease 42,000 2-pt8 Chronic rheumaticvalvular disease 42,000 2-pt9 Chronic rheumatic valvular disease 41,0003 Hepatocellular carcinoma embolus of cardiac atrium 44,000 4Hypertrophic cardiomyopathy 38,000 5 Normal great arteries tissue 37,0006 Normal cardiac atrium tissue 37,000 7 Normal myocardial tissue (focalmild hypertrophy) 38,000 8 Normal auricle of heart tissue 48,000 9Normal myocardial tissue (mild hypertrophy) 38,000 10  Normal myocardialtissue 38,000

To determine if the level of HER2 expression on cardiomyocytes issufficient to induce uptake of MM-302, total cellular doxorubicin wasquantified by HPLC following treatment of ESCd (FIG. 4A) and iPSd (FIG.4B) cardiomyocytes. Cardiomyocytes (and cancer cells) treated with freedoxorubicin result in doxorubicin accumulation in all cells. Treatmentwith PLD did not result in an increase in doxorubicin delivery in eithercardiomyocyte cell type. In contrast to HER2-overexpressing cancercells, the HER2 expression level on cardiomyocytes was not sufficient topromote active uptake of MM-302. Taken together, these resultsdemonstrate delivery of doxorubicin via MM-302 does not enhancedoxorubicin exposure to low level HER2 expressing non-target cells suchas cardiomyocytes as compared to PLD.

Example 5

MM-302 does not Reduce Human Cardiomyocyte Viability or StimulateApoptotic Responses:

Exposure to low levels of doxorubicin can be cytotoxic. To determine iftreatment with MM-302 or PLD affected cardiomyocyte viability, ESCdcardiomyocytes were incubated with free dox, PLD or MM-302 for 3 h atthe indicated concentration followed by washing and incubation in freshmedia for 24 h. Treatment with free doxorubicin resulted in a loss ofviability at concentrations as low as 0.2 μg/ml (FIG. 4C). Conversely,treatment with MM-302 and PLD did not lead to reductions in viability atany concentration tested, including super-therapeutic concentrations upto 45 μg/ml. To further test whether treatment with MM-302 or PLDaffected cardiomyocyte viability, iPSd cardiomyocytes were treated withthe indicated concentration of free doxorubicin, PLD or MM-302 for 24hours. Treatment with doxorubicin resulted in a marked decrease inviability as compared to treatment with PLD and MM-302 (FIG. 4D). Thepresence of elevated levels of cardiac troponins is a clinical indicatorof cardiac damage. The supernatant from the iPSd cardiomyocytes in (D)was analyzed for levels of troponin I. As shown in FIG. 4E, doxorubicintreatment resulted in a marked increase of Troponin I compared totreatment with PLD or MM-302. These results demonstrate that ESCd andiPSd cardiomyocytes are sensitive to doxorubicin, and that treatmentwith MM-302 and PLD does not provide sufficient doxorubicin exposure toaffect cardiomyocyte viability.

Exposure of cells to low levels of doxorubicin may induce subtlecellular changes not revealed by cell viability measurements, includingDNA damage, cell stress and incipient apoptosis. Following treatmentwith MM-302, PLD and free doxorubicin, cardiomyocytes were stained forproteins in each of these response pathways and imaged usinghigh-content microscopy. Single-cell data were generated by analyzingthe resulting images using ImageRail.

In response to double-stranded DNA damage, histone H2AX becomesphosphorylated, forming gamma-H2AX. Treatment of cardiomyocytes withfree doxorubicin resulted in a dose-dependent increase in nucleargamma-H2AX (FIG. 5A). However, treatment with MM-302 and PLD did notincrease nuclear gamma-H2AX signal at any concentration tested,indicating that liposomal encapsulation prevented DNA damage tocardiomyocytes in vitro.

In response to cellular stress, HSP27 and p53 can be phosphorylated,leading to cell cycle arrest, followed by DNA repair or apoptosisdepending on the extent of injury. Cardiac cells exposed to freedoxorubicin demonstrate a dose-dependent increase in phospho-HSP27 andphospho-p53 indicating an induction of cellular stress followingtreatment (FIGS. 5B and 5C). However, an increase in phospho-HSP27 wasnot observed in cells treated with either MM-302 or PLD regardless ofconcentration. In most cases, there did not appear to be an effect onphospho-p53 in cells treated with MM-302 or PLD, with the exception of aslight increase in phospho-p53 following treatment with 5.0 μg/ml ofMM-302. However, treatment at this and higher concentrations did notresult in increased cell death.

In cases of severe DNA damage and cell stress, the cell may initiate theapoptotic pathway including activation of a caspase cascade, ultimatelyresulting in the cleavage of the DNA repair protein PARP. Treatment with5.0 μg/ml of free doxorubicin led to an increase in nuclear cleaved PARP(cPARP) (FIG. 5D), correlating with the observed increase in cell death.However, treatment with MM-302 or PLD did not result in increasednuclear cPARP suggesting that treatment under these conditions is notsufficient to induce apoptosis.

Example 6

Impacts of HER2-Targeted Agents on Intracellular Signaling inCardiomyocytes:

The concurrent use of doxorubicin and trastuzumab is contraindicated dueto an unacceptably high incidence of cardiac events observed in patientstreated with the combination. The mechanism of action for thecardiotoxicity associated with this combination is believed to be thesimultaneous induction of cellular stress by doxorubicin and bytrastuzumab-mediated inhibition of HER2 signaling pathways that isnecessary to respond to the cellular stress induced by doxorubicin.

To determine if pretreatment with MM-302 alters HER2-mediated signaling(an essential pathway in cardiomyocytes), iPDd cardiomyocytes werepretreated for 24 hours with trastuzumab, lapatinib (a small moleculeHER2 tyrosine kinase inhibitor), or the MM-302 antibody (F5-scFv) and anempty liposome identical to MM-302 except that it does not containdoxorubicin) (F5-lipo). After stimulation with 10 nM (FIG. 6A) and 5 nM(FIG. 6B) of heregulin (HRG) for 10 min, the levels of phospho-AKT(pAKT, FIG. 6A) and phospho-ERK (pERK, FIG. 6B) were measured by highcontent microscopy as described above. Pretreatment with trastuzumab for24 h resulted in a reduction in HRG-mediated phosphorylation of bothproteins. Pretreatment with lapatinib led to a reduction in basalphosphorylation of AKT and ERK as well as a complete inhibition ofHRG-induced phosphorylation of these proteins. Pre-treatment F5-lipo didnot inhibit HRG-induced phosphorylation of AKT or ERK. These resultssuggest that, despite targeting HER2, MM-302 does not inhibitligand-induced phospho-AKT and phospho-ERK signaling in cardiomyocytes,leaving these critical signaling pathways functional.

These results also show that trastuzumab and lapatinib have asignificantly greater negative impact on this signaling pathway incardiomyocytes than do F5 scFv or F5 lipo. This in turn is an indicationthat the anti-HER2 antibody component of MM-302 is less cardiotoxic thanthe anti-HER2 antibody trastuzumab. The results show that F5, eitheralone or linked to the exterior of an MM-302 liposome, does notinterfere with heregulin (ligand)-stimulated HER2/HER3heterodimer-mediated signaling in cardiomyocytes which is an essentialintracellular signaling modality, inhibition of which is believed to bea key mechanism mediating trastuzumab-induced cardiotoxicity.

Example 7

MM-302 has a Lower Accumulation in Mouse Heart Tissue Compared to FreeDoxorubicin:

Liposome targeting with a highly specific antibody fragment such as F5generally does not alter the total tissue deposition of liposomes, butrather alters their microdistribution following extravasation. Themacro-level biodistributions of MM-302 (square), PLD (UT-PLD, triangle)and doxorubicin (free dox, circle) were compared in mouse heart tissue(FIG. 7A), human xenograft tumor tissue (FIG. 7B) and paw tissue (FIG.7C) in NCI-N87 tumor bearing mice inoculated as described above. Mice(n=4/time point/group) were injected i.v. with MM-302, PLD, or freedoxorubicin (all at 3 mg/kg dox equiv.) and hearts were collected at0.5, 4, 24, and 96 h (for dox) or 168 h (for MM-302 and PLD) postinjection and doxorubicin quantified by HPLC (FIG. 7A). Injection offree doxorubicin resulted in a high peak exposure in the heart at 0.5 h(10% of injected dose (i.d.)/g tissue) compared to the two liposomalformulations. The clearance of doxorubicin from the heart tissue afterfree doxorubicin injection was faster compared to MM-302 and PLD and at24 h the amount of detected doxorubicin (0.77% of i.d./g tissue) wasclose to background. Both MM-302 and PLD had a sustained accumulationprofile that peaked at 24 h (2.8% and 2.6% for MM-302 and PLD,respectively) while values returned to background at 168 h. Theseresults are in a range similar to that of previously reported data onthe heart biodistribution of other PLD formulations. No significantdifferences were observed between the heart biodistribution of MM-302and PLD.

Example 8 MM-302 Results in Lower Nuclear Doxorubicin Accumulation inMouse Tissue Compared to Free Doxorubicin

The microdistribution of doxorubicin (naturally fluorescent) andliposomes (DiI5-labelled) was analyzed in cryosections generated fromheart tissues of mice injected with either free doxorubicin, MM-302-DiI5or PLD-DiI5 (all at 3 mg/kg dox equiv) at 0.5, 4 and 24 h postinjection. In order to visualize the heart vasculature, mice wereinjected i.v. with FITC-lectin 5 min before sacrificing. Heart sliceswere imaged by fluorescence confocal microscopy. Representative fieldsfor the different treatment groups at the three time points analyzed(0.5, 4 and 24 h) are shown in FIG. 7D. Untreated hearts were alsoimaged and a representative image is shown in FIG. 7D (left panels).Co-localization of doxorubicin with the nuclear signal is shown in thebottom panels of FIG. 7D. Higher magnification images of the nucleardoxorubicin signal are shown in FIG. 7E, for both doxorubicin and MM-302at the 0.5 h time point. While with MM-302 no doxorubicin signal isvisible in the nuclei, with free doxorubicin the majority of the nucleiare doxorubicin-positive. The images were analyzed as described aboveand the percent of doxorubicin-positive nuclei determined. Injection offree doxorubicin resulted in a prominent nuclear accumulation ofdoxorubicin at 0.5 h, with about 50% of the nuclei positive fordoxorubicin. By 4 h, however, only 23% of the nuclei were positive fordoxorubicin and the signal returned to basal levels at 24 h.

With the liposomal formulations, little to no signal was detected forthe majority of fields of view. Occasional signal in the DiI5 channel(liposome) was detected. In these cases, the liposome signalpredominantly co-localized with the FITC-lectin signal, indicatingliposomes that had not extravasated into the heart tissue but stillremained in the vascular compartment. Upon MM-302-DiI5 or PLD-Dil5treatment, doxorubicin was not detected in the nucleus in the majorityof the heart fields analyzed, independent of time point. In one of thefour MM-302-DiI5 hearts collected at 0.5 h and in one of the fourMM-302-DiI5 hearts collected at 4 h, a liposomal signal was detected inthe extravascular space and doxorubicin was found in a small percentageof the nuclei. Similarly, in one of four PLD hearts collected at 0.5 hand in two of four PLD heart collected at 24 h, images revealed theextravascular liposomal signal and presence of nuclear doxorubicin.These fields are not presented as representative images, however theirvalues were considered for the quantification shown in FIG. 7F. The areaunder the curves of both MM-302 and PLD were statistically significantlylower than the free doxorubicin AUC (p<0.001). No significantdifferences were observed between the AUCs of MM-302 and PLD.

In order to get a broader visualization of the distribution ofdoxorubicin and of the liposomes in the heart tissue, full heart sectionscans were taken. The full section heart scans visually confirmed theresults of the confocal microscopy, showing a broad doxorubicindistribution with nuclear localization upon free doxorubicin injection,and only rare liposome and doxorubicin signals in the hearts of miceinjected with either DiI5 MM-302 or DiI5 PLD.

In summary, treatment with either MM-302 or PLD showed significantlylower nuclear doxorubicin accumulation than was seen following treatmentwith free doxorubicin, while this did not differ significantly betweenMM-302 and PLD.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain andimplement using no more than routine experimentation, many equivalentsof the specific embodiments described herein. Such equivalents areintended to be encompassed by the following claims. Any combinations ofthe embodiments disclosed in the dependent claims are contemplated to bewithin the scope of the disclosure.

INCORPORATION BY REFERENCE

The disclosure of each and every U.S. and foreign patent and pendingpatent application and publication referred to herein is specificallyincorporated by reference herein in its entirety.

What is claimed is:
 1. A method of treating a human cancer patient by administration of anthracycline-comprising anti-HER2 immunoliposomes, the method comprising determining a first dosage, such a dosage indicating a dose magnitude and frequency of dosing, for a patient diagnosed with a cancer characterized by expression of HER2 receptor, the first dosage being for a liposomal anthracycline chemotherapeutic agent that does not comprise an immunoliposome and is doxorubicin HCl liposome injection, which dosage is determined to provide to the patient a safe and effective amount of the liposomal anthracycline chemotherapeutic agent, and administering anthracycline-comprising anti-HER2 immunoliposomes, a plurality of which immunoliposomes is each bearing a plurality of anti-HER2 antibody molecules on its surface and each containing the anthracycline chemotherapeutic agent, wherein the anthracycline-comprising anti-HER2 immunoliposomes: a) are formulated in sterile 10 mM/L histidine-HCl (pH 6.5) and 10% sucrose and comprise a lipid membrane comprised of phosphatidylcholine, cholesterol, and a polyethyleneglycol-derivatized phosphatidylethanolamine in the amount of approximately one PEG molecule for 200 phospholipid molecules, of which approximately one PEG chain for each 1780 phospholipid molecules bears at its end an F5 single-chain FIT antibody fragment that binds to HER2, so that, on average, 45 copies of F5-scFv (anti-HER2) are comprised per liposome, the membrane encapsulating an aqueous space which contains 130-170 g doxorubicin/mol phospholipid, and b) are administered to the patient at the first dosage.
 2. The method of claim 1, wherein the HER2-targeted immunoliposomes are MM-302.
 3. The method of claim 1, wherein the cancer is breast cancer, Kaposi's sarcoma, ovarian cancer, or multiple myeloma.
 4. The method of claim 1, wherein the first dosage is 50 mg/m.sup.2, 40 mg/m.sup.2, 30 mg/m.sup.2, 20 mg/m.sup.2, or 10 mg/m.sup.2 every two weeks or every three weeks or every four weeks.
 5. The method of claim 1, wherein the cancer characterized by expression of HER2 receptor is further characterized as being HER2.sup.2+, HER2.sup.3+, or HER2 FISH positive.
 6. The method of claim 1, wherein the cancer characterized by expression of ErbB2 receptor further characterized as expressing an average of at least 200,000 cell surface ErbB2 receptors per cell.
 7. The method of claim 1, wherein the administration of the immunoliposomes at the first dosage is effective to treat the cancer.
 8. The method of claim 1, wherein the administration of the immunoliposomes at the first dosage does not result in increased cardiotoxicity as compared to administration at the first dosage of the liposomal anthracycline chemotherapeutic agent that does not comprise an immunoliposome.
 9. The method of claim 1, wherein the administration of the immunoliposomes to the patient at the first dosage results in a peak concentration of the immunoliposome in the patient's bloodstream and wherein treating human cardiomyocytes in vitro by culturing in medium comprising the immunoliposomes at about the peak concentration does not reduce, or reduces by no more than 5%, heregulin-stimulated increase of pERK or pAKT in the cultured cardiomyocytes as compared to in control human cardiomyocytes cultured in medium free of the immunoliposomes.
 10. The method of claim 9, wherein the immunoliposome concentration in the patient's bloodstream is measured as a serum immunoliposome concentration. 